ABSTRACT Epigenetic dysregulation is an emerging hallmark of cancers, and examples of cancer associated overexpression, mutation, translocation, or aberrant recruitment of the proteins which regulate the epigenome are rapidly emerging. NSD2 (nuclear receptor-binding SET domain-containing 2) is a key player in epigenetic regulation, known for its ability to mono- and dimethylate lysine 36 of histone 3 (H3K36). This mark is associated with active transcription, and elevated levels of H3K36me2 lead to aberrant activation of normally silenced genes. Consequently, NSD2 is a potent oncoprotein and has been implicated as a therapeutic target for a variety of cancers. NSD2 was found to be among the most frequently mutated genes across 1,000 pediatric cancer genomes representing 21 different pediatric cancer subtypes. A study in pediatric acute lymphoblastic leukemia (ALL) cell lines and patient samples revealed a recurring gain of function mutation (E1099K) which resulted in elevated H3K36me2 levels. Furthermore, NSD2 is involved in the pathogenesis of multiple myeloma (MM) which is the second most common blood cancer. 15-20% of MM patients carry a translocation between chromosomes 4 and 14 [t(4;14)(p16.3;q32)] of which NSD2 is thought to be the primary oncogenic driver. In addition to its catalytic SET domain, NSD2 contains several methyl-lysine (Kme) reader domains including four PHD fingers and two PWWP domains which are thought to be critical in propagating H3K36me2 and recruiting NSD2 to its oncogenic target genes. Although NSD2 clearly shows promise as a therapeutic target in oncology, no small molecule ligands have been reported to date and it remains unclear as to which domains are `druggable' and function most critically in promoting tumorigenesis. The creation of high-quality NSD2 chemical probes will clearly provide much needed insight and serve as critical reagents in assessing preclinical target validity, while providing the potential for an immediate transition to a drug discovery effort. Our recent efforts to target the N-terminal PWWP domain of NSD2 have been successful in producing potent lead compounds. With novel NSD2 inhibitors in hand, we aim to explore the development of NSD2 protein degradation reagents which capitalize on advantages of small molecule inhibitors while moving beyond the limitations of traditional receptor pharmacology. This emerging drug discovery strategy is particularly well-suited for NSD2 because 1) it is a large multi-domain protein and we lack knowledge of the best domain to target, 2) inhibition of a single domain may not be sufficient to phenocopy NDS2 genetic knockdown results and more broadly prevent tumorigenesis, and 3) Kme reader domains are generally viewed as difficult drug targets. The overall objective of this proposal is to apply medicinal chemistry, chemical biology, and cancer biology approaches to discover first-in-class NSD2 bifunctional degraders in order to better understand NSD2 cancer biology, to assess NSD2 preclinical target validity, and as potential therapeutic agents.